Project 1: Project Summary/Abstract Cutaneous melanomas (CM) have a very high rate of somatic mutations, and oncogenic drivers are identified in the majority of patients. PTEN, a tumor suppressor that regulates the oncogenic PI3K-AKT signaling pathway, demonstrates complete loss of expression in up to 30% of these tumors. Our previous studies showed that loss of PTEN is associated with shorter overall survival in stage III melanoma patients, and with inferior outcomes with targeted therapies in patients with stage IV disease. Building upon these studies, recently we investigated the impact of PTEN loss on the anti-tumor immune response and immunotherapy. Initial preclinical studies demonstrated that loss of PTEN in melanomas increases the expression of immunosuppressive cytokines, decreases the intratumoral infiltration of critical effector T cells, and causes resistance to T-cell mediated immunotherapy in vitro and in vivo. Analyses of cohorts of advanced melanoma patients showed that loss of PTEN was associated with decreased CD8+ T cell infiltration in stage III melanoma patients, and significantly decreased response rates to FDA approved anti-PD-1 antibodies in stage IV disease. Treatment with GSK2636771, an isoform-specific inhibitor of PI3K?, decreased AKT activation, increased T cell infiltration, and increased the efficacy of anti-PD-1 checkpoint inhibitor therapy in vivo in an immunocompetent model of PTEN-null, PD-1-resistant melanoma. Notably, GSK2636771 did not harm the viability or function of immune cells, consistent with the selective dependence on PI3K? in cells with PTEN loss. Based on these studies, we hypothesize that inhibition of the PI3K-AKT pathway will overcome resistance to anti-PD-1 immunotherapy in melanomas with loss of PTEN. To test this hypothesis, and address the unmet need for effective therapies for PD-1-refractory patients, we are conducting a phase I/II clinical trial of GSK2636771 in combination with the anti-PD-1 antibody pembrolizumab in metastatic melanoma patients with PTEN loss that failed to respond to anti-PD-1. Blood and tumor samples will be collected prior to and during treatment as well as at progression to improve our understanding of the effects of this regimen and the results of the trial. In Aim 1 we will determine the effects of this treatment on the activation of the PI3K-AKT pathway, and the relationship between pathway inhibition, GSK2636771 steady-state levels, and treatment outcomes. In Aim 2 we will evaluate the immune effects of the combination treatment by evaluating tumor and blood samples for the presence and changes in immune cell subsets and immunoregulatory cytokines, which will also be compared to clinical responsiveness. In Aim 3 we will use preclinical models to evaluate intermittent dosing and combinatorial approaches with additional isoform-selective PI3K inhibitors as strategies to further improve the efficacy of GSK2636771 with anti-PD-1. These studies will improve our understanding of the role of the PI3K-AKT pathway in the anti-tumor immune response and immunotherapy resistance, and help identify additional rational strategies for future testing in this and other cancers with PTEN loss.